1. Field of the Invention
This invention relates to the fields of drug discovery and pest control. Specifically it provides biochemical tools and assays that enable the user to identify to chemical compounds that are effective in activating or blocking particular hormone-dependent regulatory pathways in various organisms.
2. Background
Nuclear hormone receptors are a primary transduction mechanism through which extracellular hormonal signals are transduced into genetic regulation of metabolic pathways and developmental programs. The past two decades have seen the steady identification of mammalian receptors of well-known ligands such as steroids, thyroid hormone, all-trans retinoic acid (RA) as well as the identification of endogenous ligands for initially orphaned receptors. Similarly, steroid nuclear receptors in invertebrate models of transcriptional regulation, such as the Drosophila melanogaster ecdysteroid receptor (dEcR), were isolated a decade ago and used to develop important concepts in cellular hormonal signaling.
In parallel to the search for receptors that can be activated by known ligands, has been the search for ligands of orphan receptors of the steroid nuclear receptor superfamily, whose natural ligands are unknown. The biological relevance of identification of agonistic or antagonistic ligands for orphan receptors is several fold. First, the ability of a chemical structure to fit into the ligand-binding pocket of an orphan receptor and thereby transcriptionally activate the orphan receptor, would raise the possibility that the orphan receptor ligand-binding pocket has a conformation enabling it to bind with and be activated by a natural ligand of complementary structure. Second, the identification of ligands that activate or antagonize an orphan receptor would aid the discovery of regulatory pathways in which the receptor participates. Finally, transcriptional agonists and antagonists of orphan receptors provide leads to pharmacologically or agri-chemically significant structures that, through the orphan receptor, can selectively intercede in disease pathways or insect specific hormonal regulation.
In an effort to reduce the exposure of humans and other non-target organisms to the toxic effects of pesticides, major commercial efforts are being made to identify chemicals that selectively disrupt the hormonal processes that are specific to insects (e.g., humans do not have an exoskeleton that is periodically shed, nor do humans go through metamorphosis, as insects do). Two particular hormonal targets for these commercial efforts are the insect hormones that drive molting and metamorphosis: ecdysone and juvenile hormone. Some commercial compounds mimicking these hormones have already been developed, however, the concern remains that compounds interfering with insect hormonal signaling may cross-react with mammalian hormone receptors. For example, a metabolite of the juvenile hormone mimic methoprene, binds the human RXR receptor, see Dhadialla et al., Annual Review of Entomology, 43:545-569 (1998); Harmon et al. Proc Natl. Acad. Sci. USA., 92:6157-6160 (1995).
Identification of chemical compounds that bind to the ligand-binding pocket of ultraspiracle, the Drosophila RXR ortholog, has been stymied in part by difficulty in demonstrating specific binding of a test compound to the purified receptor. Specifically, it has been difficult to demonstrate ligand binding because there has been no efficient assay for detecting the conformational changes in a receptor upon interaction between a ligand and a USP and RXR. Indeed, the current paradigm expressed in most published models for USP function is that USP does not bind to any ligand in exerting its regulatory functions. Lezzi et al., (1999) Archives of Insect Biochemistry and Physiology, 41:99-106. A demonstration that endogenous USP can become activated upon binding to an agonist would have major implications for the current paradigms of hormone action in invertebrates. Jones et al., Proc Natl. Acad. Sci. USA., 94:13499-13503 (1997).
The orthology between invertebrate USP and vertebrate RXR indicates that the USP ligand-binding pocket may be arranged so as to be susceptible to binding and activation by a terpenoid-related ligand. In a previous report, it was observed that the application of methyl epoxyfarnesoate to cells activated a transfected reporter construct containing direct repeat elements to which recombinant USP bound in gel shift assay. However, these indirect experiments did not address whether methyl epoxyfarnesoate actually binds to the ligand-binding pocket of the receptor, nor whether endogenous USP in the transfected cells actually binds to the direct repeat elements, nor do they address whether methyl epoxyfarnesoate-activation of the reporter is dependent upon liganded USP, all of which are crucial underpinnings to the concept of the USP ligand-binding pocket as a viable target for experimental or practical agonistic or antagonistic ligands.